Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis

Transcription

1 The Plant Cell, Vol. 3, , May 1991 O 1991 American Society of Plant Physiologists Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis Joanne Chory,, Punita Nagpal, and Charles A. Petob a Plant Biology Laboratory, The Salk lnstitute for Biological Studies, P.O. Box 85800, San Diego, California Laboratory of Neuronal Structure and Function, The Salk lnstitute for Biological Studies, P.O. Box 85800, San Diego, California The greening phenotypes produced by recessive mutations in a gene designated de-et iolated-2 (DET2) are described. Recessive mutations in the DET2 gene uncouple light signals from a number of light-dependent processes. de12 mutations result in dark-grown Arabidopsis thaliana seedlings with many characteristics of lightgrown plants, including hypocotyl growth inhibition, cotyledon expansion, primary leaf initiation, anthocyanin accumulation, and derepression of light-regulated gene expression. In contrast to these morphological and gene expression changes, however, the chloroplast development program is not initiated in the dark in det2 mutants, suggesting that light-regulated gene expression precedes the differentiation of etioplasts to chloroplasts. det2 mutations thus reveal at least two classes of downstream light-regulated responses that differ in their timing and control mechanisms. Homozygous det 2 mutations also affect photoperiodic responses in light-grown plants, including timing of flowering, dark adaptation of gene expression, and onset of leaf senescence. The phenotype of detl det2 double mutants is additive, implying that DETl and DET2 function in distinct pathways that affect downstream light-regulated genes. Furthermore, these pathways are not utilized solely during early seedling development but must also be required to regulate different aspects of the light developmental program during later stages of vegetative growth. INTRODUCTION Seedling development proceeds according to a complex schedule of cell differentiation, cell division, and morphogenesis. This schedule includes the development and differentiation of the leaf meristem, the biogenesis of chloroplasts from undifferentiated plastids, and the regulated expression of both nuclear and chloroplast-encoded genes (Dale, 1988; Mullet, 1988; Steeves and Sussex, 1989). Dicotyledonous plants are unique in that the primary trigger for the regulation of this complex developmental program is an environmental signal, light. Dark-grown (etiolated) dicotyledonous seedlings are developmentally arrested. They have extended hypocotyls, no cotyledon expansion, no leaves, and are white. A set of well-characterized genes, the light-regulated photosynthesis genes, is not expressed or is expressed at a very low leve1 (Silverthorne and Tobin, 1987; Gilmartin et al., 1990). The transition from dark to light growth causes profound differences in the morphology of the plant, including rapid inhibition of stem elongation, expansion of cotyledons, development of To whom correspondence should be addressed leaves, and the development of the photosynthetically competent chloroplast, a process called de-etiolation (Thompson, 1988; Gruissem, 1989). During de-etiolation in Arabidopsis thaliana, for instance, the light-regulated genes become expressed at 100 to 200 times the levels at which they were expressed in the dark. The mechanisms by which light exerts its influence on this multifaceted developmental program are unknown. Both red and blue light inhibit hypocotyl elongation and induce expression of genes required for leaf and chloroplast development (Mancinelli and Rabino, 1978; Link, 1988; Thompson, 1988), whereas UV light is the strongest inducer of anthocyanin biosynthetic genes (Lipphardt et al., 1988; Schulze-Lefert et al., 1989; Staiger et al., 1989). It has been inferred that plants use at least three photoreceptors to perceive light, corresponding to red/far-red, blue, and UV wavelengths; however, the only characterized photoreceptor is the red/far-red photoreceptor phytochrome (Colbert, 1988). The number and sequence of events that occur after light excitation of the photoreceptors are not known. For instance, are there parallel pathways that regulate light-mediated seedling development?

2 446 The Plant Cell 1s there a central regulatory pathway with shared signal transduction elements that branches out into several pathways, each having an independent effect on a different aspect of light development (i.e., gene expression, leaf development, decrease in the rate of stem elongation, anthocyanin accumulation, etc.)? 1s there a linear sequence of events that occurs during seedling development? Understanding the genetic control of dicotyledonous seedling development in response to light will require identifying genes involved in directing the downstream light-regulated responses. One candidate for such a gene is the Arabidopsis DETl (de-etiolated-1) gene, which we identified during a search for mutations that induce the light developmental program in darkness (Chory et al., 1989a). detl mutants display many phenotypic characteristics of light-grown wild-type plants, even when grown in total darkness. These characteristics include leaf and chloroplast development, anthocyanin accumulation, and the accumulation of mrnas for several light-regulated nuclear and chloroplast-encoded genes (Chory et al., 1989a). Using chimeric fusion genes that were introduced into wildtype and detl lines, we have also shown that in lightgrown det 1 plants two divergent light-regulated promoters are active in cell types where they are normally silent or expressed at very low levels in wild-type plants (Chory and Peto, 1990). Taken together, these results suggest a common point, DETl, to the light-specific and tissuespecific signal transduction pathways associated with leaf and chloroplast development in Arabidopsis (Chory and Peto, 1990). The identification of mutations at the DET1 locus has been helpful in defining a potentially important signal transduction pathway involved in light-regulated early seedling development in Arabidopsis; however, these mutations have not aided the dissection of the downstream light responses (leaf development, decrease in stem elongation, chloroplast biogenesis, photoregulated gene expression). Furthermore, it is clear that additional mutations will be necessary to define the number and complexity of the pathway(s) involved in light-regulated seedling development. Here we describe the phenotype of mutations in a second gene involved in de-etiolation, DET2. Like detl mutants, det2 mutants show a deceleration of hypocotyl elongation, expanded cotyledons, anthocyanin accumulation, and increased accumulation of light-regulated nuclear and chloroplast RNAs in the absence of light. In contrast to detl, however, recessive det2 mutations do not result in the initiation of the chloroplast developmental program. Thus, det2 mutations have shown that two assayable downstream light-regulated responses (gene expression and leaf development) can be separated from the differentiation of chloroplasts. Moreover, the phenotype of det 1 det2 double mutants is additive, suggesting that two distinct pathways may be involved in the de-etiolation response in Arabidopsis. R E SU LTS lsolation and Genetic Characterization of det2 Mutants Five recessive alleles of det2 were isolated during a screen for seedlings that had a de-etiolated morphology after 7 days of growth in total darkness (see Methods). All alleles were assigned to a single locus on chromosome 2 (position 32.9) by mapping and complementation tests, as shown in Table 1 (see also Methods). All five of these lines had the same phenotype after backcrossing (see below). Three (det2-1, det2-2, and det2-3) of the five lines were backcrossed an additional three times, and the phenotypes were analyzed in detail. Figure 1 shows the dark-grown and light-grown phenotypes of det2-1 compared with wild type. After 7 days of growth in total darkness, det2 mutants were short, had thick hypocotyls, accumulated anthocyanins, and had open, expanded cotyledons (Figure 1 A). This is in contrast to wild-type dark-grown (etiolated) Table 1. Results of Crosses with det2-1 Mutant Cross TvDe Total De-etiolated Etiolated y2 a detl -1 ldetl-1 37 o 37 det2-1 ldet2-1 det2-1 ldet2-1 DET2IDET2 DET2/det 2-1 DET2ldet2-1 DET2/det2-1 det2-1 ldet2-1 DET2/det 2-1 DET2pet2-2 DET2/det2-1 DET2/det2-3 DET2/det2-1 DET2/det2-4 DET2/det2-1 DET2/det2-5 F, 55 o FP Testcross a x2 calculation for an expected segregation of one de-etiolated to three etiolated.

3 Light-Regulated Development in Arabidopsis 447 seedlings, which had long hypocotyls and folded cotyledons and did not accumulate anthocyanins (Figure 1A). If kept in the dark for longer periods of time, det2 alleles developed primary leaf buds, although no further leaf development was observed (data not shown). Wild-type etiolated seedlings never developed further than shown in Figure 1A. The det2 homozygotes also had an abnormal phenotype when grown in the light (Figure 1B). They were smaller and darker green than wild-type plants grown under the same conditions. In addition, det2 mutants had a prolonged vegetative phase (see below). det2 mutants made at least twice the number of leaves as wild type grown under the same light regimes (an average of 19 ± 3 leaves/ plant, compared with 9 ± 2 leaves/plant for wild type), and had reduced apical dominance (average of five to seven bolts/plant for det2, whereas wild type had one to two bolts/plant). Finally, homozygous det2 alleles had smaller flowers than wild type and all five alleles had severely reduced male fertility. The significance of this increased male sterility is not known. Nuclear and Chloroplast Light-Regulated Genes Are Constitutively Expressed in the Dark in det2 Plants in the Absence of Chloroplast Development Figure 1. Phenotypes of Dark-Grown and Light-Grown del 2 and Wild-Type Arabidopsis Seedlings. (A) Seven-day-old dark-grown (etiolated) wild type (left) and 7- day-old dark-grown ctef2 (right). (B) Twelve-day-old light-grown wild type (left), and 12-day-old light-grown det2 (right). Bar = 1cm. We examined the accumulation of mrnas known to be positively regulated by light in wild-type and det2 seedlings grown in the light and dark. These included both nuclear and Chloroplast genes for chloroplast-localized proteins, as well as a blue-light-regulated nuclear gene encoding chalcone synthase (ens) (Feinbaum and Ausubel, 1988), which encodes an anthocyanin biosynthetic enzyme. The mrnas examined included those corresponding to the nuclear genes for the small subunit of ribulose bisphosphate carboxylase, rbcs (Krebbers et al., 1988), the genes for the light-harvesting chlorophyll a/b-binding proteins, cab (Leutwiler et al., 1986), and four different chloroplastencoded mrnas: psaa-b, which codes for the 66-kD chlorophyll apoproteins of photosystem I; psba, the 32- kd Q B -binding protein of photosystem II; rbcl, the large subunit of ribulose-bisphosphate carboxylase; and 16S rrna (Mullet and Klein, 1987). In all of the nuclear and Chloroplast photosynthesis genes that we examined, we observed a high level of mrna accumulation in dark-grown det2 seedlings. Several are shown in Figure 2. These levels were 10-fold to 20-fold higher than those in darkgrown wild-type seedlings (Figure 2) and were about 10% to 20% of wild-type light-grown levels. In the case of the ens gene, the mrna levels in dark-grown det2 seedlings were at least 50-fold higher than wild-type etiolated seedlings and were about half of the levels accumulated in wildtype light-grown plants.

4 448 The Plant Cell rbcs cab psba rdna Figure 2. Accumulation of mrnas for rbcs and cab (Nuclear Genes) and psba (a Representative Chloroplast Gene) in Light- Grown and Dark-Grown Wild-Type and def2 Plants. Lane 1, light-grown wild type; lane 2, dark-grown wild type; lane 3, light-grown det2; lane 4, dark-grown det2. An Arabidopsis rdna probe was used to normalize the load in the various lanes. Three micrograms of total RNA was loaded per lane. Similar results were observed for other chloroplast-encoded genes (rtocl, psaa-b) as for psba (data not shown). Table 2 depicts the average GUS levels observed in wildtype and det2 transgenic seedlings either grown in the light or germinated and grown in darkness. In dark-grown ctef2 seedlings, the levels of GUS activity from the cab3 promoter were about 10% of the levels in light-grown det2 seedlings (Table 2). This 10-fold light induction contrasts with the greater than 80-fold level of light induction observed in wild-type plants. In chs-gus transgenic def2 seedlings, the chs promoter drove GUS expression to greater than 5 times the levels in dark-grown wild-type seedlings, which corresponded to approximately one-third of the light-grown c/is-gus activity (Table 2). These data indicate that, in addition to the high levels of RNA accumulated, there was also aberrant high constitutive transcription from these promoters in dark-grown ctef2 seedlings. def2 mutants accumulated high levels of mrnas for photosynthesis genes in the dark. To determine whether the proteins translated by these RNAs were also accumulated in dark-grown det2 seedlings, we examined the accumulation of several photosynthetic proteins using antibodies specific for purified proteins or complexes. The same plants that were used for the RNA gel blot analyses were used for these studies. A representative protein gel blot for light-harvesting chlorophyll a/b proteins (LHCPII) (which are coded for by the cab genes) and the ribulosebisphosphate carboxylase/oxygenase (Rubisco) (coded for by rbcs and rbcl) is shown in Figure 3. For Rubisco, aberrant high levels of both the small and large subunits were observed in dark-grown defl and def 2 mutants (about 15% of wild-type light levels) (Figure 3B, lanes WT, detl, and det2). This contrasts to dark-grown wild-type seedlings where no Rubisco proteins were observed (Figure 3B, lane et). In the case of LHCPII, no protein accumulated in either dark-grown def 1 or det2 seedlings, even though high levels of cab mrnas were present in these seedlings. This result was not surprising because neither We previously showed, for def 1 mutants, that the aberrant dark expression of nuclear light-regulated genes is correlated with increased transcription from these promoters (Chory and Peto, 1990). Similarly, we analyzed the def2 mutant for aberrant transcriptional regulation using def2 mutant lines carrying a transgenic Escherichia coli /3-glucuronidase (GUS) marker gene under the control of the blue-light-regulated chs promoter (Feinbaum and Ausubel, 1988; Chory and Peto, 1990) or the phytochromeregulated cat>3 promoter (Chory and Peto, 1990). The cab-gus and c/is-gus fusions were introduced into def2 mutants by crossing with well-characterized wild-type transgenic lines (see Methods) (Chory and Peto, 1990). Table 2. Light-Dark Expression of cab3 and chs Promoters in Wild-Type and det2 Seedlings Construct cab3-gus crts-gus GUS Units 8 Wild Type Light 16,500 34,600 Dark L/D b det2 Light 1 1,900 29,700 Dark L/D b Plants were grown for 14 days in the light (light) or were germinated in the light overnight and then grown in the dark for 7 days (dark). 8 GUS units are picomoles of 4-methylumbelliferone per minute per milligram of protein. b Ratio of light versus dark GUS units.

5 Light-Regulated Development In Arabidopsis 449 WT et CD CM «CD a large paracrystalline body, the prolamellar body, and the notable absence of thylakoid membranes. ctef2 plastids were also etioplasts (Figure 4B). No chloroplasts were ever observed in dark-grown det2 plants. det2 Mutants Have a Phenotype in the Light, Characterized by a Prolonged Juvenile Phase and a Lack of Leaf Senescence B Figure 3. Immunoblot Analyses of the Levels of LHCPII or Rubisco Proteins Accumulated in Dark-Grown ctefl, det2, and Wild- Type Seedlings Compared with Light-Grown Wild Type. Lane WT, wild type grown for 14 days in the light; lane et, wild type grown for 7 days in total darkness (etiolated); lane detl, def\ grown for 7 days in total darkness; lane det2, det2 grown for 7 days in total darkness. LSU and SSU refer to the large and small subunits of the Rubisco, respectively. Fifty micrograms of protein was loaded per lane. ctem nor cter2 accumulates chlorophylls when grown in the dark. Previous work from several laboratories has shown that the light-harvesting chlorophyll apoproteins are rapidly turned over in the absence of chlorophylls (Apel, 1979; Bennett, 1981), and we assume this is the basis for the observation here. We examined plastid morphology in dark-grown der2 and wild-type seedlings. Figure 4 shows that the chloroplast developmental program has not been initiated, even though there are cotyledon expansion, aberrant high levels of nuclear and chloroplast gene expression, and accumulation of Rubisco in dark-grown ctef2 mutants. Etiolated wild-type seedlings have a plastid form, the etioplast, that is typified by the structure shown in Figure 4A; these plastid types were characterized by their irregular shape, Plants possess distinct juvenile and adult developmental phases. The juvenile or vegetative phase is the period of growth during which no flowering is possible (Hart, 1988). One of the most important factors that affects the transition from vegetative to floral growth is the environmental signal, light; however, other factors such as nutrition, temperature, and unknown endogenous factors are also involved (Hart, 1988). In Arabidopsis, the length of the juvenile phase can vary greatly, with flowering possible as early as the cotyledonary stage if the plants are stressed, or delayed for several months if the daylength is short. However, if the growth conditions are well defined, the vegetative growth phase is actually quite predictable. Wild-type Arabidopsis (ecotype Columbia) seedlings, grown in a 22 C growth chamber on a 16-hr day/8-hr night growth regime, germinated in approximately 3 days and produced a rosette of 9 ± 2 leaves. Plants made the transition from the vegetative to floral growth phases approximately 21 days postgermination. This transition was characterized by the rapid growth of the floral blot and was followed by leaf senescence (beginning at about 30 days). During leaf senescence, chloroplast volume and number per mesophyll cell declined, resulting in the concomitant decrease in chlorophyll concentration and causing the plants to yellow. Light-regulated genes coding for photosynthetic functions were expressed at relatively low levels during senescence, as shown in Figure 5, lanes 4 and 5. By the time of seed set (approximately 7 weeks), the rosette leaves were brown and atrophied. ctef2 mutants, in contrast, had a prolonged vegetative growth phase. Under the same growth conditions just described for wild-type plants, det2 mutants produced a rosette of 19 ± 3 leaves, had small leaves that resembled young leaves, and did not flower until 33 days postgermination. Moreover, no visible leaf senescence was observed even after 7 weeks of growth. To assess whether other light-regulated functions associated with greening were affected, we prepared samples for RNA and chloroplast analysis from wild type and ctef2. Figure 5 shows an autoradiograph of an RNA gel blot of cab mrna accumulation with time. Wild-type leaves showed a dramatic (10-fold) decline of cab mrna by 22 days of growth in light; however, def2 plants had high levels of cab mrna even after 45 days of growth. The results were reinforced by measurements of the specific chlorophyll content in these plants with time, as described in Table 3. Together,

6 «*s/ >.-"^ L. ^* ti i /.7. B ^ ^ Figure 4. Electron Micrographs of Representative Plastids from Dark-Grown Wild-Type and det2 Seedlings. (A) Wild-type etioplast. (B) det2 etioplast. Bar = 1 t^m.

7 Light-Regulated Development in Arabidopsis 451 WILD-TYPE det Figure 5. Accumulation of cab mrnas during Development in Wild Type and det2. Lanes 1, 8 days postgermination; lanes 2, 14 days postgermination; lanes 3, 19 days postgermination; lanes 4, 22 days postgermination; lanes 5, 45 days postgermination. Five micrograms of total RNA was loaded per lane. these results implied that the chloroplasts were still present in high numbers and functionally active in det2 plants, even after 7 weeks of growth in the light. We followed the time course of chloroplast development in wild-type versus det2 plants. Eight-day-old wild-type chloroplasts, shown in Figure 6A, had the typical lens shape and granal stacking of a mature chloroplast. Eightday-old det2 chloroplasts (Figure 6E), in contrast, resembled immature leaf chloroplasts, with a smaller, rounder shape and reduced granal stacking. Furthermore, 8-dayold det2 seedlings had an abnormally high ratio of chlorophyll a/b, indicating the relatively "young" state of the chloroplasts (Table 3). By day 14, the det2 chloroplasts were similar morphologically to wild-type chloroplasts and continued to grow and divide through day 22 (Figure 6G). Wild-type chloroplasts looked similar to the 8-day-old chloroplasts until the first flowers were observed (Figure 6D) at approximately day 22. Shortly after that time, visible signs of leaf senescence had occurred, and we did not take any more samples for microscopy. Figure 61 shows a typical det2 chloroplast after 45 days of growth in the light. In general, the chloroplasts were still intact; however, it appeared that although chloroplast replication events had occurred, the daughter chloroplast remained fused to the mother. Also, in sections, the grana membranes had a novel circular swirling appearance. The membrane stacks were distorted, with many individual stacks curved or bent, instead of oriented somewhat parallel to the outer chloroplast membrane and inclusions. We had not observed this phenomenon previously. det2 Mutations Affect the Expression of Nuclear Light-Regulated Genes during Light to Dark Transitions Because the ctef2 mutations affected the photoperiodic control of vegetative development, we were interested to see whether the DET2 gene product was also necessary for the proper day-night-regulated expression of photosynthesis genes, such as cab. We addressed this question by analyzing RNA from det2 and wild-type plants grown in continuous light for 3 weeks and then shifted to the dark for 24 hr or 48 hr. It has been shown previously that accumulation of RNA for nuclear genes involved in photosynthesis decreases in the dark (Giuliano et al., 1988; Chory et al., 1989a). When we examined the levels of accumulation of cab mrnas in dark-adapted ctef2 mutants, we observed different results than for wild type, as shown in Figure 7. In wild type, the levels of cab RNA had decreased by approximately eightfold after 24 hr in the dark and were undetectable by 48 hr; however, in def2, cab RNA levels had decreased by only about twofold to threefold after 24 hr and 12-fold after 48 hr (Figure 7). Similar results were observed for rbcs RNAs (data not shown). These experiments suggest that the DET2 gene product may be involved in a signaling pathway that affects the expression of cab and rbcs genes during light-dark transitions. det 1 def 2 Double Mutant Studies We generated plant lines homozygous for both ctem and def 2 mutations to examine the epistatic relations of these two genes and possible phenotypic interactions. The light and dark phenotypes of the double mutants indicate that the combination of two homozygous det mutations in a Table 3. Chlorophyll Content and Chlorophyll a/b Ratio of Wild Type and det2 during Development sowing Chlorophyll Content" (/ag/seedhng) Wild Type det Chlorophyll a/b Ratin a Wild Type def " Values are an average of three seedlings for two independent experiments. On days 28 and 45, flowering bolts were removed.

8 Figure 6. Electron Micrographs of Representative Chloroplasts during Development in Wild Type and ctef2. (A) Wild type, 8 days postgermination. (B) Wild type, 14 days postgermination. (C) Wild type, 19 days postgermination. (D) Wild type, 22 days postgermination. (E) det2, 8 days postgermination. (F) def2, 14 days postgermination. (G) def2 19 days postgermination. (H) def2, 22 days postgermination. (I) detz, 45 days postgermination. Bar = 1 /im.

9 Light-Regulated Development in Arabidopsis Wild-type I^F W* det Figure 7. Accumulation of cab RNA in Dark-Adapted Wild-Type and det2 Plants. Lanes 1, 15-day-old seedlings grown in continuous light; lanes 2, 15-day-old seedlings grown in continuous light and shifted to total darkness for 24 hr; lanes 3, 15-day-old seedlings grown in continuous light and shifted to total darkness for 48 hr. Two micrograms of RNA was loaded per lane. single plant results in an additive interaction. Similar results were observed using two different alleles of ctef2. The light-grown phenotypes for each mutant parent and for the double mutant are shown in Figure 8 and summarized in Table 4. Each individual det mutation affected different light-regulated processes in the light, and upon examination of some of these traits, a simple additive phenotype was observed in the double mutants (Table 4). Although the def 1 ctef2 double mutants make the vegetative to floral transition (Figure 8D), bolts never elongated, and we have been unable to propagate the double mutants by either selfing or crossing with the wild-type or individual mutant parent lines. DISCUSSION We have shown that recessive mutations in the DEJ2 gene uncouple light signals from a number of light-dependent processes. def2 mutations result in dark-grown seedlings with many characteristics of light-grown wild-type seedlings, including inhibition of the rate of hypocotyl elongation, cotyledon expansion, development of primary leaf buds, accumulation of anthocyanins, and derepression of light-regulated gene expression. Although det2 mutations affect many downstream processes of seedling development that are normally light regulated, one notable exception is the lack of differentiation of etioplasts to chloroplasts. Thus, mutations in the DET2 gene have helped in the dissection of some of the downstream light-regulated processes. At the very least, these studies have shown that regulation of genes for chloroplast-destined proteins must precede any morphological differentiation of etioplasts to chloroplasts. This characteristic distinguishes DET2 from D 7~1 because in dem mutants, all assayable downstream light-regulated responses are affected, including leaf development, chloroplast development, and gene expression (Chory et al. 1989a). de(2 mutants also have an aberrant phenotype when grown in the light; therefore, it is likely that DET2 plays some role in light-grown plants. The most striking characteristics of the phenotype of light-grown def2 plants are reduced leaf growth, a prolonged vegetative phase, reduced male fertility, and a lack of leaf senescence, even after flowering. The leaves of def2 mutants remain green after seed set, and we never observed drastic diminishing of chloroplast numbers or breakdown of granal membranes over a 7-week growth period. This prolonged juvenile phase was characterized by high levels of cab mrna accumulation throughout vegetative growth, which is atypical of wild-type plants. Normally, cab mrna levels are highest during early seedling growth and diminish during later stages of vegetative growth (e.g., much higher levels at 7 days versus 3 weeks in Arabidopsis). Heterochronic mutations (Tec-pod) affecting maize shoot development have been described (Poethig, 1988). These three semidominant mutations have a profound effect on both vegetative and reproductive development. Although it is difficult to compare maize to Arabidopsis directly, because of morphological differences, certain characteristics of the Jp mutants and the def2 mutants are similar. For instance, the Jp mutants have an increased number of vegetative phytomers, an increased number of phytomers producing ears, a decrease in the size of leaves and internodes, and a decrease in the size of the ear and tassel (Poethig, 1988). Plants homozygous for the def2 mutation bear similar phenotypic characteristics. There are, however, several notable differences between the Jp mutants and def 2. For instance, Jp mutations are semidominant and probably represent gain-of-function mutations, whereas all five def2 alleles are completely recessive and have the same phenotype, implying that each allele may represent the null state of the gene. Also, the Jp mutants have a homeotic transformation of reproductive structures into vegetative ones, and we never observed any signs of organ conversion in det2 mutants. The timing of flowering was also delayed in def 2 mutants (by at least 10 days versus the wild type), and thus def2 could be classified as a "late-flowering" mutant. Although the molecular mechanisms involved in floral induction are unknown, physiological studies implicate roles for light (both photoperiod and quality), temperature, and assimilate availability in the timing of floral induction (e.g., Martinez- Zapater and Somerville, 1990). \narabidopsis, several lateflowering mutants have been identified (Koornneef et al., 1983) and a subset of these has been further characterized

10 B D Figure 8. Photographs of Wild Type, Single Mutants, and Double Mutant. (A) Twenty six-day-old wild-type plant. (B) Fifty-day-old def2-1 plant. (C) Thirty-five-day-old dem-1 plant. (D) Forty two-day-old def1-1 def2-1 double mutant. defl def2-2 double mutants had the same phenotype as the seedling shown in (D). Bar = 1 cm.

11 ~~ ~ ~~~ Light-Regulated Development in Arabidopsis 455 Table 4. Summarv of detl det2 Double Mutant Phenotvoe Genotype Phenotype Dark Light No. Days to Roots Leaves Pigmentation Flower Wild type Etiolated White 9 Green 21 detl De-etiolated Green 1 O Pale green 21 det2 De-etiolated White 19 Dark green 33 detl det2 De-etiolated Green 12 Green 28 (Martinez-Zapater and Somerville, 1990). Studies have not been performed on the greening responses of these mutants. It would be interesting to test whether the prolonged juvenile phase of these mutants is marked by persistent cab gene expression and chloroplast development, as reported here for the det2 mutants. A third characteristic of the light-grown phenotype of det2 mutants is the slower kinetics of the decline of cab and rbcs mrna after the transfer of light-grown plants to darkness for 1 day to 2 days. Although the implications of these results are currently unclear, more detailed experiments should be performed to evaluate this phenomenon. Homozygous det2 mutations are pleiotropic, affecting the switch between etiolation and de-etiolation, as well as affecting photoperiodic responses, including timing of flowering, dark adaptation of cab gene expression, and delay of leaf senescence. Given the recessive nature of the mutations and the phenotype of the mutants, one prediction is that DET2 is a component in a signal transduction pathway whose activity is high during etiolation, low during early seedling development (de-etiolation), and relatively high during later vegetative growth. Thus, loss-of-function mutations in DET2 result in de-etiolated seedlings in the dark and a prolonged vegetative growth in the light, and DET2 may be a molecular switch involved in the decisionmaking process between etiolation and de-etiolation in Arabidopsis. The DET2 switch would be used throughout development to allow plants to progress into an adult phase when chloroplast replication and maintenance are no longer crucial. A second alternative is that det2 mutants define a developmental pathway whose activity is modulated by light. A possible candidate would be a cytokinincontrolled pathway involved in the regulation of leaf development and senescence. DET2 is the second genetic locus we have described that appears to be involved in a signal transduction pathway that regulates the etiolation-de-etiolation response. det2 mutations share some similarities with detl mutations, yet there is a distinct set of light-regulated processes that are affected differently in detl versus det2 mutants. The summary of the dark-grown and light-grown phenotypes of detl and det2 mutants is shown in Table 5. Both DETl and DET2 appear to be involved in light signal transmission because mutations at either locus uncouple a number of light-dependent processes from the light signal. Mutations in the DETl gene result in derepression of the entire light developmental program, including leaf and chloroplast development and gene expression (Table 5) (Chory et al., 1989a). Furthermore, detl mutants are defective in the proper integration of temporal or spatial regulatory signals because we observed a loss of tissuespecific expression of the chloroplast developmental program and the light-regulated gene expression program, including aberrant expression of cab promoters in roots Table 5. Comparison of der1 and det2 Mutants Wild Tvpe det 1 det2 Dark phenotype Morphology Leaves Unexpanded Expanded cotyledons Expanded cotyledons cotyledons and leaves (primary leaf buds) Hypocotyl Long Short Short Pigments Absent Anthocyanins Anthocyanins Chloroplasts Undifferentiated Some differentiation Undifferentiated Gene expression (% of wild-type levels in the light) Nuclear O Chloroplast O0 10 Light phenotype Morphology No. of leaves 9k2 10r Bolt no Days to flower Tissue-specific gene expression affected No Yes No Light-dark regulation affected No No Yes

12 456 The Plant Cell and the chs promoter in leaf mesophyll cells and flowers (Chory and Peto, 1990). Thus, DETl may be involved in the integration of light with tissue-specific signals that affect greening in Arabidopsis. In general, der2 mutations are less severe than detl mutations. det2 mutations affect some of the downstream light-regulated processes, including morphology (cotyledon expansion, hypocotyl growth deceleration, and leaf development) and gene expression. However, we saw no effect on the etioplast to chloroplast transition in darkgrown seedlings. Furthermore, unlike detl mutants, det2 mutants never make rosettes in the dark; only primary leaf buds were observed. In addition, although light-regulated genes were expressed at 1 O-fold to 20-fold higher levels in dark-grown det2 mutants than wild-type levels in the dark, these levels were still about 10-fold lower than lightgrown plants and fivefold to 1 O-fold lower than der 1 plants grown in the dark. We recently proposed that the extent of light-regulated gene expression was dependent on the amount of leaf development (Chory et al., 1989a). The characterization of gene expression patterns in darkgrown det2 mutant seedlings reinforces this hypothesis. The extent of leaf development is light-grown wild-type plants > dark-grown detl plants > dark-grown det2 plants > wild-type etiolated seedlings. When the levels of lightregulated gene expression were measured for the above, we obtained the same relationship described for leaf development. Furthermore, based on the phenotype of det2 mutants, chloroplast development does not appear to be required for derepression of the expression of nuclear and chloroplast-encoded photosynthesis genes. Interestingly, both nuclear and chloroplast mrnas accumulated to about the same levels in dark-grown det2 seedlings, indicating that the nuclear and chloroplast compartments were coordinately affected by det2 mutations. Nuclear and chloroplast compartments were similarly affected by detl mutations as well (Chory et al., 1989a). detl and det2 mutations appear to affect a different subset of light-regulated functions in light-grown plants (see Table 5). Therefore, perhaps it is not surprising that detl det2 double mutants have a much more severe phenotype than either single mutation alone. Although we do not have direct proof that the detl and det2 alleles are null, the nearly additive interaction observed between the detl and det2 mutations suggests an absence of interaction between the two gene products. Therefore, it seems likely that eitherdet1 and DET2 do not lie within a common signal transduction pathway or they act in separate branches of a common signal transduction pathway. Moreover, we have evidence that both det 1 and der2 mutations are epistatic to severa1 different hy mutations that result in phytochrome deficiency in Arabidopsis (J. Chory, unpublished results), and the most probable order of gene action would place both DETl and DET2 downstream of phytochrome activation. Thus, the identification of detl and der2 mutants has allowed us to propose that at least two distinct pathways are involved in the etiolation-de-etiolation response in Arabidopsis. Furthermore, these pathways are not utilized solely during early seedling development but must also be required to regulate different aspects of the light developmental program during vegetative development. We are currently in the process of cloning DETl and DE T2 so that the molecular mechanisms of lightmediated seedling development can be unraveled. METHODS Plant Material, Growth Conditions, and Genetic Methods Two det mutants were isolated originally afier ethyl methane sulfonate mutagenesis of wild-type seeds from the Columbia ecotype (Chory et al., 1989a). We mutagenized an additional 20,000 Ml seeds and isolated three additional independent det2 alleles (Table 1). Each mutant was backcrossed into the wild-type Columbia background before complementation analysis was performed; three alleles were backcrossed an additional three times before the physiological studies. AI1 five of these lines have the same phenotype after backcrossing (see Results). In all the experiments described here, the wild type was the Columbia line. Genetic nomenclature is based on the Third lnternational Arabidopsis Meeting (East Lansing, MI, 1987). If not specified, it is assumed that the mutant allele number is 1 (e.g. det2-1 is referred to as det2). Each mutant phenotype segregated as a single recessive nuclear mutation in genetic crosses (see Table 1 for data on det2-1). Because of the almost complete mate sterility of the det2 mutant lines, it was necessary to maintain these lines as heterozygotes. Fl plants were generated after crossing the original deetiolated mutant (M2) with wild-type pollen. The seeds were collected from each F1 plant and used as a source of segregating homozygous mutant plants. The mutants were identified by germination on synthetic medium plus sucrose in the dark and scoring the de-etiolated phenotype in the Fp generation (see Table 1). For complementation studies, the recovery of de-etiolated plants among the progeny of a cross between two heterozygotes at a ratio of one de-etiolated to three etiolated (wild type) indicated noncomplementation (Table 1 ). These results indicated that all five mutations lie in the same complementation group. To obtain the chromosomal location of the det2 mutation, an ecotype Landsberg marker line that contained one visible marker on chromosomes 1 (an), 3 (g/-l), 4 (cer-2), and 5 (ms-l), and two markers on chromosome 2 (er, position and py, position 2.22) was used (see Koornneef, 1990, for map positions). The ms-1 marker was maintained in the heterozygous state (+/ms-1). Pollen from the Landsberg tester strain was crossed to det2-1, and det2 Fp plants were chosen and scored for segregation of the visible markers. Additional markers on chromosome 2 used for linkage analysis were hyl (position ) (Koornneef, 1990), hy6 (position ) (Chory et al., 1989b; J. Chory and L. Altschmied, unpublished data), and a previously mapped kan' transgene from a transformation experiment (position ) (L. Altschmied and J. Chory, unpublished data). det2 was mapped to chromosome 2, position 32.9 (data not shown). We have not resolved the map position of det2 relative to the as marker on

13 Light-Regulated Development in Arabidopsis 457 chromosome 2, which maps to position 33. Using restriction fragment length polymorphism probes from chromosome 2, we have confirmed that det2 is tightly linked to as (data not shown; Nam et al., 1989). Plants were grown at 20 C under a mixture of fluorescent and incandescent lights at an intensity of 300 pe/m'/sec. Methods for the growth of plants in pots, seed harvesting, and cross-pollination have been described (Somerville and Ogren, 1982). For all the experiments on light-grown plants, det2-1, det2-2, det2-3, and wild type were grown side-by-side under the same light and humidity conditions for the times indicated in the text. Dark-grown seeds were germinated for 36 hr in the light on synthetic medium plus sucrose and then transferred to total darkness for 7 days, as previously described (Chory et al., 1989a). Germination of seedlings by this protocol gave the same results as when the seedlings were germinated directly in the dark with gibberellic acid in the medium (data not shown). RNA Gel Blots RNA was isolated from small amounts of tissue by using the acid guanidium method of Chomczynski and Sacchi (1987). RNA (2 pg to 5 pg) was separated in formaldehyde-containing agarose gels (Ausubel et al., 1987) and blotted onto Nytran (Schleicher & Schuell) using the manufacturer's recommendations. After transfer to the nylon membrane, the RNA was cross-linked to the membrane by UV irradiation (Stratalinker). 32P-labeled DNA probes (5 x 10' cpm/wg) were generated by random primer extension (Feinberg and Vogelstein, 1983). The filters were hybridized (5 x 106 cpm/ml) using the conditions of Church and Gilbert (1984). Filters were washed three times at 60 C in 0.2 x SSC, 1.O% SDS for 20 min each, and exposed to preflashed Kodak AR-5 film at -7OOC with an intensifying screen. To normalize for RNA loading, filters were stripped and rehybridized with an rdna probe. Autoradiograms for different exposure times were scanned with a densitometer. Relative amounts of mrnas were determined by peak-area measurements, and relative mrna levels reported are an average of two separate hybridizations. The DNA probes for nuclear and chloroplast genes used in these studies were as published elsewhere (Chory et al., 1989a). Analysis of Chimeric Transgenes in Wild Type and det2 The cab-gus and chs-gus fusions were transformed into wildtype Columbia as described elsewhere (Chory and Peto, 1990). For the experiments described here, the cab-gus and chs-gus fusions were introduced into the det2 mutant by crossing with wild-type transgenic lines. Kan' F, seedlings were allowed to selfpollinate, and kan' det2 mutants were screened in the Fp progeny and used for subsequent analysis. Electrophoresis and lmmunological Detection of Proteins Seven-day-old dark-grown and 20-day-old light-grown Arabidopsis seedlings were quickly frozen and ground with a mortar and pestle in liquid nitrogen. The samples were then transferred to a lyophilization apparatus without thawing. The lyophilized tissue was extracted into SDS extraction buffer (0.5 ml of buffer/30 mg of powder), heated to 7OoC for 1 O min, and electrophoresed in a 12% SDS-polyacrylamide gel (Laemmli, 1970). The amount- of protein loaded was 50 pg per lane. Electrophoretic transfer of protein to nitrocellulose, reaction with antisera, and anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad lmmunoblot kit) were performed as described by the manufacturer. The relative levels of specific proteins were quantified by scanning photographic negatives with a densitometer. The antibody probes used in these studies were rabbit anti-lhcpii (pea), obtained from Dr. J. Bennett (Bennett et al., 1984) and rabbit anti-rubisco (tobacco large subunit, pea small subunit), obtained from Dr. S. Rodermel (Rodermel et al., 1988). Electron Microscopy For dark-grown wild type and det2, cotyledon tissue from 7-dayold seedlings grown in absolute darkness [on solidified synthetic Murashige and Skoog (1962) medium supplemented with 1% sucrose] was used as the source of material. For light-grown seedlings, leaf samples were harvested at 8 days, 14 days, 17 days, and 22 days from both wild type and det2 that had been grown side-by-side under the same light and humidity conditions. For det2, an additional time point after 45 days in the light was also collected. Tissues were fixed with glutaraldehyde followed by osmium tetroxide, dehydrated, and embedded in Spurr resin. Analytical Techniques Chlorophyll determinations were performed on leaves and stems from wild-type and det2 plants harvested after 8 days, 14 days, 17 days, 22 days, and 45 days of growth in the light and immediately frozen in liquid nitrogen. The frozen tissue was later ground in liquid nitrogen in a mortar and pestle, and chlorophyll was extracted repeatedly into 80% acetone in the dark until the pellet appeared colorless. Chlorophyll a and b contents were calculated using MacKinney's specific absorption coefficients (MacKinney, 1941), in which chlorophyll a = 12.7 (A663) (Asd5) and chlorophyll b = 22.9(&45) (&63). The total specific chlorophyll Content is expressed as micrograms of chlorophyll per seedling. GUS assays with protein extracts of leaves and stems were carried out using the fluorometric assay described by Jefferson (1987). Protein determinations were made with a Bio-Rad protein assay kit. ACKNOWLEDGMENTS J.C. thanks Nathan Aguilar for assistance with some experiments. We thank Stephen Worland, Ron Susek, Terry Delaney, and Alan Pepper for comments on the manuscript, and Paul Sawchenko for generous use of electron microscope facilities. This research was supported by grants from the National Science Foundation and the Department of Energy (to J.C.) and by The Samuel Roberts Noble Foundation. Marc Lieberman of The Salk lnstitute for Biological Studies prepared the cover photograph and Figures 1 and 8. Received January 22, 1991; accepted March 6, 1991.

14 458 The Plant Cell REFERENCES Apel, K. (1979). Phytochrome induced appearance of mrna activity for the apoprotein of the light-harvesting chlorophyll a/b protein of barley. Eur. J. Biochem. 97, Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1987). Current Protocols in Molecular Biology. (New York: Green Publishing Assoc./Wiley Interscience) Bennett, J. (1981). Biosynthesis of the light-harvesting chlorophyll a/b protein. Polypeptide turnover in darkness. Eur. J. Biochem. 118, Bennett, J., Jenkins, G.I., and Hartley, M.R. (1984). Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves. J. Cell. Biochem. 25, Chomczynski, P., and Sacchi, N. (1987). Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform-extraction. Anal. Biochem. 162, Chory, J., and Peto, C. (1990). Mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and chloroplast development in Arabidopsis. Proc. Natl. Acad. Sci. USA 87, Chory, J., Peto, C., Feinbaum, R., Pratt, L., and Ausubel, F. (1989a). Arabidopsis fhaliana mutant that develops as a lightgrown plant in the absence of light. Cell. 58, Chory, J., Peto, C.A., Ashbaugh, M., Saganich, R., Pratt, L., and Ausubel, F. (1989b). Different roles for phytochrome in etiolated and green plants deduced from characterization of Arabidopsis fhaliana mutants. Plant Cell 1, Church, G.M., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, Colbert, J.T. (1988). Molecular biology of phytochrome. Plant Cell Environ. 11, Dale, J.E. (1988). The control of leaf expansion. Annu. Rev. Plant. Physiol. Plant MOI. Biol. 39, Feinbaum, R., and Ausubel, F.M. (1988). Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. MOI. Cell. Biol. 8, Feinberg, A.P., and Vogelstein, 6. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, Gilmartin, P. M., Sarokin, L., Memelink, J., and Chua, N.-H. (1990). Molecular light switches for plant genes. Plant Cell 2, Giuliano, G., Pichersky, E., Malik, V.S., Timko, M.P., Scolnik, P.A., and Cashmore, A.R. (1988). An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene. Proc. Natl. Acad. Sci. USA 85, Gruissem, W. (1989). Chloroplast gene expression: How plants turn their plastids on. Cell 56, Hart, J.W. (1988). Topics in Plant Physiology. (London: Unwin Hyman Ltd.). Jefferson, R.A. (1987). Assaying chimeric genes in plants: The GUS gene fusion system. Plant MOI. Biol. Rep. 5, Koornneef, M. (1990). Linkage map of Arabidopsis fhaliana. In Genetic Maps: Locus Maps of Complex Genomes, 5th ed., S.J. O'Brien, ed (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp Koornneef, M., van Eden, J., Hanhard, C.J., Stam, P., Braaksma, F.K., and Feenstra, W.J. (1983). Linkage map of Arabidopsis fhaliana. J. Hered. 74, Krebbers, E., Seurinck, J., Herdies, L., Cashmore, A.R., and Timko, M.P. (1988). Four genes in two diverged subfamilies encode the ribulose-l,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana. Plant MOI. Biol. 11, Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, Leutwiler, L.S., Meyerowitr, E.M., and Tobin, E.M. (1986). Structure and expression of three light-harvesting chlorophyll a/b binding protein genes in Arabidopsis fhaliana. Nucl. Acids Res. 14, Link, G. (1988). Photocontrol of plastid gene expression. Plant Cell Environ. 11, Lipphardt, S., Brettschneider, R., Kreuzaler, F., Schell, J., and Dangl, J.L. (1988). UV-inducible transient expression in parsley protoplasts identifies regulatory cis-elements of a chimeric Anfirrhinum majus chalcone synthase gene. EMBO J. 7, MacKinney, G. (1 941). Absorption of light by chlorophyll solutions. J. Biol. Chem. 140, Mancinelli, A.L., and Rabino, 1. (1978). The high irradiance responses of plant photomorphogenesis. Bot. Rev. 44, Martinez-Zapater, J.M., and Somerville, C.R. (1990). Effect of light quality and vernalization on late flowering mutants of Arabidopsis thaliana. Plant Physiol. 92, Mullet, J.E. (1 988). Chloroplast development and gene expression. Annu. Rev. Plant Physiol. Plant MOI. Biol. 39, Mullet, J.E., and Klein, R.R. (1987). Transcription and RNA stability are important determinants of higher plant chloroplast RNA levels. EMBO J. 6, Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, Nam, H.-G., Giraudat, J., den Boer, B., Moonan, F., Loos, W.D.B., Hauge, B.M., and Goodman, H.M. (1989). Restriction fragment length polymorphism linkage map of Arabidopsis fhaliana. Plant Cell 1, Poethig, R.S. (1988). Heterochronic mutations affecting shoot development in maize. Genetics 119, Rodermel, S.R., Abbot, M.S., and Bogorad, L. (1988). Nuclearorganelle interactions: Nuclear antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in transformed tobacco plants. Cell 55, Schulre-Lefert, P., Dangl, J.L., Becker-Andre, M., Hahlbrock, K., and Schulr, W. (1989). lnducible in vivo DNA footprints